There are the 14 known mammalian isoforms of carbonic anhydrases. Each of these isozymes possesses carbon dioxide-hydrating activity that functions to maintain intracellular pH. Carbonic anhydrase isozyme III (CAIII) is distinguished from the other isoforms by several characteristics--particularly, by a low specific activity that is about 1 percent that of isozyme II. CAIII is not inhibited by acetozolamide, an excellent inhibitor of CAI and CAII. Also, the amount of CAIII is remarkably high in muscle tissue and adipocytes, constituting about 8 and 25 percent of the soluble protein content of these tissues. Despite numerous publications on the enzyme, the physiological function of CAIII remains unknown. We created a knockout to facilitate investigation of the function of CAIII. The genomic DNA of mouse CAIII was cloned by a colony plaque hybridization method. Using a positive/negative-type targeting vector, about 4.1 kb of CAIII genomic DNA, including the exons 3, 4, and 5, was replaced by the bacterial neomycin (neo) resistance gene driven by the phosphoglycerate kinase 1 promoter. The knockout vector was initially electroporated into the embryonic stem cells. G418 was used to select cells containing the plasmid and gancyclovir was used to select against cells in which the construct had integrated randomly. Resistant colonies were then grown to confluence, replicated, and genomic DNA from one set was analyzed by a polymerase chain reaction (PCR) method. Eight correctly targeted clones were selected among the 118 double positive clones. Several embryonic cell lines carrying the mutant CAIII allele were injected into host blastocysts, and nine chimeras were born and tested for transmission of the mutation to their offspring. Screening through both PCR and Southern blotting showed that the heterozygotic mice (F1) lacked one of the alleles of CAIII. To test for possible embryonic or fetal wastage, heterozygotic F1 mutant mice were mated. The homozygous (F2) animals are viable with the oldest currently aged 11 months. Thus far, no phenotype has been observed in these F2 homozygotes. Offspring of heterozygote matings were genotyped. The results were clearly Mendelian. We have experimentally tested two hypotheses for the physiological function of CAIII. The first was that CAIII conferred resistance to oxidative stress. This was tested by exposing mice to 100 percent oxygen, which revealed no difference in resistance among wild type, heterozygote, and homozygote knockouts. The second hypothesis suggested that CAIII was essential for normal fat tissue maintenance, since it constitutes one-third of the soluble protein in adipocytes. However, fat distribution and amounts were normal in the knockouts. Experiments now in progress are aimed at assessing other possible roles of CAIII, especially in muscle.